A useful assaying technique using stem-loop oligonucleotide probes referred to as “molecular beacons” was first disclosed as providing a rapid, quantitative assay technique by Tyagi and Kramer (Tyagi, S., F. R. Nature Biotechnology, 14, 303-308 (1996)). Molecular beacons are designed to have loop sequences which are complementary to a target nucleic acid (e.g., rRNA). The loop sequence is disposed between a first and a second stem sequence, the respective stem sequences being complements of one another. The molecular beacon includes a fluorescent molecule on the end of the first stem and a quenching molecule on the end of the second stem.
In the absence of the complementary target sequence the fluorescence upon irradiation remains low (quenched) due to physical proximity between the fluorophore and the quencher. When the complementary target sequence is present, the loop opens and the fluorophore and the quencher are no longer in physical proximity, so that the molecular probe generates a relatively strong fluorescent signal upon irradiation when the target nucleic acid sequence is present.
Because of their inherent signal transduction mechanism, molecular beacons (MBs) have many advantages over traditional DNA probes, including enhanced specificity and sensitively and the ability to detect target without separation of hybridized and non-hybridized probes. This detect-without-separation make MBs useful in situations where it is not possible or desirable to isolate the probe-target hybrids from an excess of the unhybridized probes, such as in the real time monitoring of polymerase chain reactions in sealed tubes, or in the monitoring of mRNAs in living cells.
Since the first report of the MB in 1996 by Tyagi and Kramer, great efforts have been made to improve the MB designs. The targets of the MBs have also been extended from original DNA or RNA molecules to now include a variety of protein molecules. Molecular beacons have become one of the important tools in the field of molecular biology studies, clinical diagnostics as well as biotechnologies.
The applications and potential of molecular probes relying on fluorescence resonance energy transfer (FRET) to detect and report binding to target molecules in general have been hindered by low sensitivity. Theoretically, molecular beacons should have up to 200 times of enhancement in fluorescence signal over traditional DNA probes. However, this enhancement has rarely been achieved in molecular beacon applications. Low signal enhancement is believed to the result of many factors, including formation of secondary structures, sticky end pairing, presence of impurities and the low quenching efficiency of the quencher molecule in the molecular probe. Among them, the former two factors could be eliminated by careful design of the probe sequences. The latter two factors are major sources of the background signal.